The essentially 100% frequency of mutational activation of the KRAS oncogene in pancreatic cancers (PDAC), together with the strong validated role of aberrant KRAS function in pancreatic cancer development and tumor maintenance, argues that anti-Ras inhibitors will provide a very effective therapy for PDAC. Perhaps the most promising avenues for anti-Ras inhibitors are antagonists of Ras effector signaling. However, these efforts have been complicated by the multitude of effector pathways that may promote Ras-mediated oncogenesis. Our recent studies validated the RalGEF-Ral small GTPase pathway as a critical mediator of KRAS-dependent PDAC growth. We therefore hypothesize that inhibition of Ral signaling will provide effective inhibition of PDAC growth. Our identification of two distinct pharmacologic approaches for blocking RalGEF-Ral signaling, inhibitors of geranylgeranyltransferase-I and Aurora-A protein kinase posttranslational modification of Ral, establishes two exciting possibilities to accomplish this. Therefore, the main objectives of our proposed studies will be to perform further preclinical evaluation of these directions, including studies on state-of-the art human primary xenografts and KRAS driven genetically-engineered mouse models, to further investigate the mechanisms of RalGEF-Ral activation in PDAC growth, and to identify additional directions for blocking Ral function in PDAC. We propose four Specific Aims to [1] determine if inhibition of geranylgeranyltransferase-I (GGTI-2417) and Ral GTPase membrane association are sufficient to inhibit pancreatic tumorigenesis and metastasis, [2] determine if the anti-tumor activity of Aurora-A kinase inhibitors (MP529 and MLN8237) requires the inhibition of RalA function and whether additional protein kinases regulate Ral GTPase function, [3] determine whether GGTase-I and/or Aurora-A inhibition (alone or in combination with gemcitabine) are sufficient to impair the growth of patient-derived primary pancreatic xenografts and pancreatic tumorigenesis and metastasis in a KRAS-driven mouse model, and [4] identify clinically relevant transcriptional targets of Ral GTPase activation, as diagnostic or therapeutic targets, of RalA- and RalB-mediated oncogenesis. Our long-term goal is to transition anti-Ral approaches into Phase I/II clinical trials for PDAC PUBLIC HEALTH RELEVANCE: Pancreatic cancer remains one of the deadliest cancers. We have shown that the RalGEF-Ral pathway is clinically and functionally important in pancreatic cancer. The goals of this proposal are to study the RalGEF- Ral pathway as a therapeutic target for pancreatic cancer treatment by using both available pharmacologic approaches and by identifying novel approaches to target this pathway.